Fabrication of Micropatterned Self-Oscillating Polymer Brush for Direction Control of Chemical Waves.

The propagation control of chemical waves via a pentagonal patterned structure in a self-oscillating polymer brush composed of N-isopropylacrylamide and a metal catalyst for the Belousov-Zhabotinsky (BZ) reaction is reported. The patterned self-oscillating polymer brush is prepared by combining surface-initiated atom transfer radical polymerization and maskless photolithography. Surface modification is confirmed by X-ray photoelectron spectroscopy, Fourier transform infrared spectroscopy, 3D measuring laser microscopy, and fluorescence microscopy. The polymer brush patterns are fabricated with gaps between the pentagonal regions, and investigations on the effect of the gap distance on the BZ reaction reveal that at the appropriate distance, chemical waves propagate across the array from the plane to the corner between the patterns. Unidirectional control is achieved not only in the 1D array, but also in a 2D curved array. This patterned self-oscillating polymer brush is a novel and advantageous approach for creating an autonomous dynamic soft interface.

Human Neural Tissue Construct Fabrication Based on Scaffold-Free Tissue Engineering.

Current neural tissue engineering strategies involve the development and application of neural tissue constructs produced by using an anisotropic polymeric scaffold. This study reports a scaffold-free method of tissue engineering to create a tubular neural tissue construct containing unidirectional neuron bundles. The surface patterning of a thermoresponsive culture substrate and a coculture system of neurons with patterned astrocytes can provide an anisotropic structure and easy handling of the neural tissue construct without the use of a scaffold. Furthermore, using a gelatin gel-coated plunger, the neuron bundles can be laid out in the same direction at regulated intervals within multilayered astrocyte sheets. Since the 3D tissue construct is composed only by neurons and astrocytes, they can communicate physiologically without obstruction of a scaffold. The medical benefits of scaffold-free tissue generation provide new opportunities for the development of human cell-based tissue models required to better understand the mechanisms of neurodegenerative diseases. Therefore, this new tissue engineering approach may be useful to establish a technology for regenerative medicine and drug discovery using the patient's own neurons.

Controlling shape and position of vascular formation in engineered tissues by arbitrary assembly of endothelial cells.

Cellular self-assembly based on cell-to-cell communication is a well-known tissue organizing process in living bodies. Hence, integrating cellular self-assembly processes into tissue engineering is a promising approach to fabricate well-organized functional tissues. In this research, we investigated the capability of endothelial cells (ECs) to control shape and position of vascular formation using arbitral-assembling techniques in three-dimensional engineered tissues. To quantify the degree of migration of ECs in endothelial network formation, image correlation analysis was conducted. Positive correlation between the original positions of arbitrarily assembled ECs and the positions of formed endothelial networks indicated the potential for controlling shape and position of vascular formations in engineered tissues. To demonstrate the feasibility of controlling vascular formations, engineered tissues with vascular networks in triangle and circle patterns were made. The technique reported here employs cellular self-assembly for tissue engineering and is expected to provide fundamental beneficial methods to supply various functional tissues for drug screening and regenerative medicine.

The mechanism of melanocytes-specific cytotoxicity induced by phenol compounds having a prooxidant effect, relating to the appearance of leukoderma.

Specific phenol compounds including rhododendrol (RD), a skin-brightening ingredient in cosmetics, are reported to induce leukoderma, inducing a social problem, and the elucidation of mechanism of leukoderma is strongly demanded. This study investigated the relationship among the cytotoxicities of six phenol compounds on B16F10 melanoma cells and HaCaT keratinocytes and generated reactive oxygen species (ROS). As a result, the cytotoxicity of RD on B16F10 cells was higher than that on HaCaT cells, and RD significantly increased intracellular ROS and hydrogen peroxide (H2O2) levels in B16F10 cells. Furthermore, although raspberry ketone (RK), RD derivative, also increased intracellular ROS in B16F10 cells, increase in ROS was suppressed by disodium dihydrogen ethylenediaminetetraacetate dehydrate (EDTA). The amounts of increased ROS with RK in HaCaT cells without melanocyte were further increased by tyrosinase. Therefore, tyrosinase, a metalloprotein having copper, was speculated to be one of causative agents allowing phenol compounds to work as a prooxidant. Hydroxyl radical was generated by adding a mixture of tyrosinase and H2O2 to RD, and the amount of the radical was further increased by UVB, indicating that RD cytotoxicity was caused by intracellularly increased ROS, which possibly related to phenol induced prooxidants.

Stripe-patterned thermo-responsive cell culture dish for cell separation without cell labeling.

A stripe-patterned thermo-responsive surface is prepared to enable cell separation without labeling. The thermo-responsive surface containing a 3 µm striped pattern exhibits various cell adhesion and detachment properties. A mixture of three cell types is separated on the patterned surface based on their distinct cell-adhesion properties, and the composition of the cells is analyzed by flow cytometry.

Micropatterning with a liquid crystal display (LCD) projector.

Photolithography has been applied to biological applications such as cell and protein micropatterning and the fabrication of microfluidic channels. However, the preparation of photomasks for projecting micropattern lighting images is often time consuming and costly. Therefore, we have developed maskless photolithography devices by modifying the optics of commercially available liquid crystal display (LCD) projectors from extended to reduced projection. The developed second and third devices produce practically a centimeter-scale micropattern by dividing an original large mask pattern into several patterns, which are individually and synchronously exposed to substrates with a motorized XY-stage, applying them to cell micropatterning and polydimethylsiloxane (PDMS) microfluidic device production. The first part of this chapter describes the developments of the maskless photolithography devices. The second part describes the exposure control system with a motorized XY-stage. The third part describes the applications of devices to cell micropatterning. The last part describes the application of the devices to the fabrication of the PDMS microfluidic channel. Maskless photolithography with an LCD projector has a large advantage with no requirement for a photomask. In particular, the maskless photolithography devices show a greater power by optimizing the conditions of pattern size and shape.

Rate control of cell sheet recovery by incorporating hydrophilic pattern in thermoresponsive cell culture dish.

Thready stripe-polyacrylamide (PAAm) pattern was fabricated on a thermoresponsive poly(N-isopropylacrylamide) (PIPAAm) surface, and their surface properties were characterized. A PIPAAm surface spin-coated with positive photoresist was irradiated through a 5 µm/5 µm or a 10 µm/10-µm black and white striped photomask, resulting in the radical polymerization of AAm on the photoirradiated area. After staining with Alexa488 bovine serum albumin, the stripe-patterned surface was clearly observed and the patterned surface was also observed by a phase contrast image of an atomic force microscope. NIH-3T3 (3T3) single cells were able to be cultured at 37°C on the patterned surfaces as well as on a PIPAAm surface without pattern, and the detachment of adhered cells was more rapidly from the patterned surface after reducing temperature. Furthermore, the rate of detachment of 3T3 confluent cell sheet on the patterned surface was accelerated, compared with on a conventional PIPAAm surface under the static condition. The rate control of cell sheet recovery should contribute the preservations of cell phenotype and biological functions of cell sheet for applying to clinical trials.

Semi-circular microgrooves to observe active movements of individual Navicula pavillardii cells.

We performed a trajectory analysis of movements of Navicula pavillardii diatom cells that were confined to semi-circular microgrooves with several different curvature radii. Using the semi-circular micropattern, we succeeded in observing change of velocity of the same cell before and after the stimulation by N,N-dimethyl-p-toluidine (DMT). Because the looped grooves had longer contour length than straight grooves, it was effective to achieve the long term observation of the stimulated active cells. Although average velocity of 150 cells was significantly increased with DMT, the maximum velocity (19 µm/s) of the cells was not increased after the DMT injection. This may suggest that existence of the mechanical limit of the velocity of the diatom cells. Secondly, trajectories of individual cell movements along the walls of the semi-circular microgrooves were analyzed in detail. As a result, the velocity of the cells was not affected by the curvature radii of the grooves although the trajectories indicated an obvious restriction of area of the cell motion. This suggests that the surface of the diatom is effective in minimizing the frictional force between the cell body and the wall of a groove. Finally, a simple model of cell motion in the semi-circular groove was proposed to clarify the relationships among the forces that determine cell movement.

Control of the formation of vascular networks in 3D tissue engineered constructs.

Construction of bio-mimetic well-organized three-dimensional (3D) tissue with various cells in vitro is one of the ultimate goals of tissue engineering. In particular, fabrication of vasculature in 3D tissue is one of the most important tasks in tissue engineering, because a vascular network is indispensable for almost every tissue in our body. Here, we sandwiched stripe patterned endothelial cells by randomly cultured fibroblast sheets to control the formation of vasculature in the tissue. The endothelial cells left the original pattern and formed a random network between the two sheets, but, where fibroblasts were focally oriented, some endothelial cells changed their orientation to the same direction as the surrounding fibroblasts. Based on this phenomenon, we sandwiched stripe-patterned endothelial cells between parallel-oriented fibroblast sheets to construct a continuous pre-vascular structure. In the tissue, endothelial cells maintained the shape of their original pattern. On the other hand, stripe-patterned endothelial cells that were vertically sandwiched between oriented fibroblast sheets diverged from the original pattern at right angles, so that they were aligned with the surrounding fibroblasts. These data indicates that, 3D design with consideration of cell-to-cell interaction is critical to fabricate a specific 3D tissue structure. The 3D-designed tissue will become a powerful tool for the study of pharmacology and biology, the substitution of animal models and the fabrication of vascularized tissue grafts.

Shear stress-dependent cell detachment from temperature-responsive cell culture surfaces in a microfluidic device.

A new approach to quantitatively estimate the interaction between cells and material has been proposed by using a microfluidic system, which was made of poly(dimethylsiloxane) (PDMS) chip bonding on a temperature-responsive cell culture surface consisted of poly(N-isopropylacrylamide) (PIPAAm) grafted tissue culture polystyrene (TCPS) (PIPAAm-TCPS) having five parallel test channels for cell culture. This construction allows concurrently generating five different shear forces to apply to cells in individual microchannels having various resistance of each channel and simultaneously gives an identical cell incubation condition to all test channels. NIH/3T3 mouse fibroblast cells (MFCs) and bovine aortic endothelial cells (BAECs) were well adhered and spread on all channels of PIPAAm-TCPS at 37 °C. In our previous study, reducing culture temperature below the lower critical solution temperature (LCST) of PIPAAm (32 °C), cells detach themselves from hydrated PIPAAm grafted surfaces spontaneously. In this study, cell detachment process from hydrated PIPAAm-TCPS was promoted by shear forces applied to cells in microchannels. Shear stress-dependent cell detachment process from PIPAAm-TCPS was evaluated at various shear stresses. Either MFCs or BAECs in the microchannel with the strongest shear stress were found to be detached from the substrate more quickly than those in other microchannels. A cell transformation rate constant C(t) and an intrinsic cell detachment rate constant k(0) were obtained through studying the effect of shear stress on cell detachment with a peeling model. The proposed device and quantitative analysis could be used to assess the possible interaction between cells and PIPAAm layer with a potential application to design a cell sheet culture surface for tissue engineering.

Hippo pathway regulation by cell morphology and stress fibers.

The Hippo signaling pathway plays an important role in regulation of cell proliferation. Cell density regulates the Hippo pathway in cultured cells; however, the mechanism by which cells detect density remains unclear. In this study, we demonstrated that changes in cell morphology are a key factor. Morphological manipulation of single cells without cell-cell contact resulted in flat spread or round compact cells with nuclear or cytoplasmic Yap, respectively. Stress fibers increased in response to expanded cell areas, and F-actin regulated Yap downstream of cell morphology. Cell morphology- and F-actin-regulated phosphorylation of Yap, and the effects of F-actin were suppressed by modulation of Lats. Our results suggest that cell morphology is an important factor in the regulation of the Hippo pathway, which is mediated by stress fibers consisting of F-actin acting upstream of, or on Lats, and that cells can detect density through their resulting morphology. This cell morphology (stress-fiber)-mediated mechanism probably cooperates with a cell-cell contact (adhesion)-mediated mechanism involving the Hippo pathway to achieve density-dependent control of cell proliferation.

Micropatterned thermoresponsive polymer brush surfaces for fabricating cell sheets with well-controlled orientational structures.

Newly developed fabrication technique of thermoresponsive surface using RAFT-mediated block copolymerization and photolithography achieved stripe-like micropatterning of poly(N-isopropylacrylamide) (PIPAAm) brush domains and poly(N-isopropylacrylamide)-b-poly(N-acryloylmorpholine) domains. Normal human dermal fibroblasts were aligned on the physicochemically patterned surfaces simply by one-pot cell seeding. Fluorescence images showed the well-controlled orientation of actin fibers and fibronectin in the confluent cell layers with associated extracellular matrix (ECM) on the surfaces. Furthermore, the aligned cells were harvested as a tissue-like cellular monolayer, called "cell sheet" only by reducing temperature below PIPAAm's lower critical solution temperature (LCST) to 20 °C. The cell sheet harvested from the micropatterned surface possessed a different shrinking rate between vertical and parallel sides of the cell alignment (approximately 3:1 of aspect ratio). This indicates that the cell sheet maintains the alignment of cells and related ECM proteins, promising to show the mechanical and biological aspects of cell sheets harvested from the functionalized thermoresponsive surfaces.

The co-application effects of fullerene and ascorbic acid on UV-B irradiated mouse skin.

The role of fullerene as a pro-oxidant or anti-oxidant in Ultraviolet B ray (UV-B)-induced disorders in mouse skin was investigated. Fullerene gave no photo-toxic effect to UV-B-irradiated mouse skin. Since erythema was concentrated at the pore circumference in a UV-B irradiation experiment in mouse skin, the sebaceous gland pairs was strongly implicated as a site for the generation of reactive oxygen species (ROS). In a histological evaluation of the skin stained with CH(3)MDFDA (ROS index) and YO-Pro-1 (apoptosis index), the fluorescence intensity of a sebaceous gland significantly increased with UV-B irradiation. With the application of fullerene to UV-irradiated mouse skin, no toxicity was recognized in comparison with the control, and erythema, the ROS index, and the apoptosis index decrease with the application of fullerene. Ascorbyl radical (AA*) increased with the application of ascorbate (AA) to UV-B-irradiated mouse skin, and AA* decreased with the application of fullerene. The co-application of AA and fullerene, which suppressed AA* in vitro, significantly suppressed erythema, and also suppressed both the ROS index and apoptosis index in mouse skin after UV-B irradiation. In both mouse skin at 48 h after UV-B irradiation and in an attempt to reproduce this phenomenon artificially in vitro, a similar high AA* peak (AA*/H*>4) was observed in electron spin resonance (ESR) charts. The binding of fullerene with AA impairs the Fenton reaction between AA and Fe-protein based on the observation of ascorbate-specific UV absorption and a linear equation for the calibration curve. Therefore, fullerene may impair the intercalation of AA to a heme pocket by binding with AA. These results suggest that the co-application of AA and fullerene is effective against oxidative skin damage caused by UV-B irradiation, and the development of an AA* inhibitor such as fullerene should be useful for reducing organ damage associated with Fe-protein oxidation.

Fabrication of transferable micropatterned-co-cultured cell sheets with microcontact printing.

The purpose of the present study is to develop a novel method for the fabrication of transferable micropatterned cell sheets for tissue engineering. To achieve this development, microcontact printing of fibronectin on commercially available temperature-responsive dishes was employed. Primary rat hepatocytes were seeded on the dish surfaces printed with fibronectin. Under serum-free conditions, hepatocytes were attached onto fibronectin domains selectively. Then, a second cell type of endothelial cells was seeded in the presence of serum. Double fluorescent staining revealed that endothelial cells successfully adhered to the intervals of hepatocyte domains. Finally, all the cells were harvested as a single contiguous micropatterned cell sheet upon temperature-reduction. With a cell sheet manipulator having a gelatin layer for the support of harvested cell sheets, harvested micropatterned cell sheets were transferred to new dish surfaces. This technique would be useful for the fabrication of thick tissue constructs having a complex microarchitecture.

Mass preparation of size-controlled mouse embryonic stem cell aggregates and induction of cardiac differentiation by cell patterning method.

Embryonic stem cells (ESCs) are promising cell sources for cell-based therapy. It has been established that the formation of ESC aggregates promotes their differentiation into the derivatives of all three germ layers. ESC aggregates are generally prepared via the formation of suspended spherical aggregates called embryoid bodies (EBs). Because the differentiation efficiency depends on the size of EBs, it becomes one of the research topics how to prepare size-controlled EBs in a scalable manner for reproducible and high-throughput experiments. Here, we have developed a novel culture method that enables simple mass preparation of size-controlled ESC aggregates on a culture surface instead of floating EBs. We developed a maskless photolithography device that enabled rapid fabrication of micropatterned surfaces. Utilizing this device, we fabricated the culture substrates the surfaces of which comprised arrays of cell-adhesive circular micro-domains (100-400 microm in diameter) and the rest of non-cell-adhesive domains. We seeded mouse ESCs on this substrate and prepared size-controlled ESC aggregates on the micro-domains. We analyzed cardiac differentiation in the ESC aggregates and found that the optimal diameter of micro-domains was 200 microm. The present method is useful for the simple and reproducible mass preparation of ESC-derived differentiated cells and high-throughput assays.

[Cell sheet engineering--fabrication and utilization of patterned cell sheet].

Tissue engineering seeks to provide regenerated tissue architectures in vitro but has not yet successfully created thick, highly vascularized, multi-functional tissues replicating native structure. We have proposed cell sheet engineering to overcome the problems of tissue engineering. And we have focused attention on micropatterned structure of cells in tissue. Therefore we have developed maskless photolithography devices equipped with LCD-projectors to fabricate micropatterned surfaces rapidly and easily. In this paper, we introduce the fabrication technique of cell micropatterns with the maskless photolithography device. And we describe our recent approaches to fabricate vascularized tissue equivalents using cell sheet engineering and the cell micropatterning technique.

Second-generation maskless photolithography device for surface micropatterning and microfluidic channel fabrication.

We have previously reported on a maskless photolithography device for surface micropatterning and microfabrication by modifying a commercially available liquid crystal display projector. For the prototype, 10-microm resolution was achieved by downsizing the image on a 0.7-in. liquid crystal display panel to an area of 8 x 6 mm and projecting it on a fixed stage. Here, we report on a second-generation maskless photolithography device having two novel features. First, the sliding lens system with variable focal distances and exchangeable objective lenses achieves a variable resolution of 2-8 mum. Second, the synchronous control of displayed images generated by a personal computer and the movement of a XY-positioning stage allows for the fabrication of micropatterns over a larger area (over 50 x 50 mm). Here, we show examples fabricated with the two novel features.

Maskless liquid-crystal-display projection photolithography for improved design flexibility of cellular micropatterns.

We previously developed an all-in-one photopolymerization device by modifying a commercially available liquid crystal display projector (LCDP) for the preparation of micropatterned surfaces and microfluidic channels without the need for expensive photomasks. In the present study, we demonstrate a simple and reliable method for rapid prototyping of cell micropatterning with high resolution using the modified LCDP device. Fabrication of complicated and flexible patterns was achieved using this device with positive-type photoresist in a two-step process. First, micropatterns on the silanized coverslips were fabricated from positive photoresist. Second, acrylamide monomer solution containing polymerization initiator was dropped onto the micropatterned positive photoresist and copolymerized on the silanized coverslips in situ by thermally initiated radical polymerization. After the reaction, the remnant micropatterned photoresist is easily dissolved, resulting in a polyacrylamide-silane micropattern on the coverslip. The resultant polyacrylamide layer is highly hydrophilic and repels both protein adsorption and cell adhesion. Cells seeded on the micropatterned surfaces therefore attach and spread only on unpolymerized silanized glass surfaces, conforming to the pattern design. This technique is therefore useful for inexpensive, rapid prototyping of surface micropatterns using polymer materials.

Micropatterned surfaces prepared using a liquid crystal projector-modified photopolymerization device and microfluidics.

A commercial liquid crystal device projector was modified for photopolymerization using its on-board intense light source and a precision optical control circuit. This device projects reduced images generated by a typical personal computer onto the stage where photopolymerization on a surface occurs. This all-in-one device does not require expensive photomasks and external light sources. However, light scattering and diffraction through glass substrates resulted in undesired reactions in areas corresponding to masked (black) domains in mask patterns, limiting pattern resolution. To overcome this shortcoming, two-step surface patterning was developed. First, three-dimensional microstructures of crosslinked silicone elastomer were fabricated with this device and adhered onto silanized glass substrate surfaces, forming microchannels in patterns on the glass support. Then, acrylamide monomer solution containing photoreactive initiator was flowed into these micromold channels and reacted in situ. The resultant polyacrylamide layer was highly hydrophilic and repelled protein adsorption. Cell seeding on these patterns in serum-supplemented culture medium produced cells selectively adhered to different patterns: cells attached and spread only on unpolymerized silanized glass surfaces, not on the photopolymerized acrylamide surfaces. This technique should prove useful for inexpensive, rapid prototyping of surface micropatterns from polymer materials.

Cell micropatterning using photopolymerization with a liquid crystal device commercial projector.

Photopolymerization has been widely used for surface micropatterning. The technique often requires photomasks and light sources with appropriate energies or filters. For rapid prototyping of surface photo-micropatterning, we have developed a novel device by modifying a commercially available liquid crystal device projector. In place of the image expansion unit of the projector, we attached an image reduction unit, an adjustable stage, and an optical monitoring unit. The device projected computer-generated images onto surfaces and subjected these patterns to photopolymerization. Micropatterned images can be easily prepared with various software run on personal computers. With the developed photopolymerization device, micropatterning of poly(ethylene glycol) (PEG) was achieved with PEG-diacrylate and a visible light photopolymerization initiator, camphorquinone. Selective cell adhesion control was also achieved on the micropatterned surfaces.